Vectors and methods for enhanced cell longevity and protein expression

ABSTRACT

It is the object of the current invention to provide methods and compositions relating to the expression of vankyrin proteins in cell lines to increase their viability, longevity and capacity for protein production. The inventors have discovered that the expression of P-ank-1 and I 2 -ank-3 proteins in cell culture has increased the cells&#39; longevity and capacity for endogenous and/or heterologous target protein production. Specifically, the present invention relates to the enhanced expression of endogenous and/or heterologous target proteins/polypeptides in recombinant cells that are also expressing P-ank-1 and/or I 2 -ank-3 protein compared to expression host cells that are not expressing P-ank-1 and/or I 2 -ank-3 protein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the fields of nucleic acid constructs and celllines that allow for the increased expression of endogenous orheterologous target protein.

2. Background

The immediate challenge created by the genomics era is the production ofthe novel proteins to understand their function. Current methods ofexpressing genes in a mammalian cell include the use of viral vectors,such as those which are derived from retroviruses, adenoviruses, herpesviruses, vaccinia viruses, polio viruses, sindbis viruses, oradeno-associated viruses. Other methods of expressing an exogenous genein a mammalian cell include direct injection of DNA, the use ofligand-DNA conjugates, the use of adenovirus-ligand-DNA conjugates,calcium phosphate precipitation, and methods which utilize a liposome-or polycation-DNA complex.

Due to its advantages in versatility and speed, the BaculovirusExpression Vector System (BEVS) used in conjunction with insect cellshas become well-established for the production of proteins, particularlyrecombinant glycoproteins. Baculovirus mediated protein expressionprovides correct folding of recombinant protein as well as disulfidebond formation, oligomerization and other important post-translationalmodifications that impart proper biological activity and function. Withregard to protein folding and post-translational processing, insectcells are second only to mammalian cell lines when expressing aeukaryotic protein, for example. The frequent use of baculovirus arisesfrom the relative ease and speed with which a heterologous protein canbe expressed on the laboratory scale and the high chance of obtaining abiologically active protein. Insect cells can be grown on serum freemedia which is an advantage in terms of costs as well as of biosafety.For large scale culture, conditions have been developed which meet thespecial requirements of insect cells.

In nature, baculoviruses are double-stranded DNA-containing viruses thatinfect a variety of different insect species. The nuclear polyhedrosisviruses, which comprise subgroup A of the Family Baculoviridae, inducethe formation of paracrystalline occlusion bodies in the nuclei ofinfected host cells. These occlusion bodies are composed primarily of asingle viral protein which is expressed at very high levels(polyhedrin). In later stages of the infection cycle, polyhedrin mayaccount for more than 50% of the total protein in an infected cell. Thepolyhedrin gene has been cloned and sequenced and its unique featureshave provided the basis for the development of a series of baculovirusexpression vectors (BEVs: Summers, M. D. and Smith, G. E., TAES Bull.1555 (1987); Luckow, V. A. and Summers, M. D., Biotechnology 6:47-55(1988); Miller, L. K., Ann. Rev. Microbiol. 42:177-179 (1988); U.S. Pat.No. 4,745,051, G. E. Smith and M. D. Summers (Filed May 27, 1983; IssuedMay 17, 1988)).

BEVs are recombinant baculoviruses in which the coding sequence forpolyhedrin has been replaced with the coding sequence for a desiredprotein. In general, this approach involves the construction andisolation of recombinant baculoviruses in which the coding sequence forthe chosen gene has been inserted behind the promoter for thenonessential polyhedrin viral gene (Pennica, et al, Mol. Cell. Biol.4:399-406 (1984); Smith, et al, L. Virol. 46:584-593 (1983); Smith, G.E. and M. D. Summers, Mol. Cell. Biol. 3:2156-2165 (1983). Severaladvantages may exist when employing the BEV system. One of theseadvantages is the strong polyhedrin promoter which directs a high levelof expression of the inserted heterologous nucleic acid encoding thetarget polypeptide. The newly expressed heterologous target proteinaccumulates in large amounts within these infected insect cells. Thus,as a result of the relative strength of the polyhedrin promoter, manydifferent gene inserts can be expressed at very high levels.

In addition to providing a high expression level, another advantage ofthe BEV system is the ease with which these baculoviruses are producedand identified. This process begins by co-transfecting wild-type viralDNA and a “transfer vector” into susceptible host cells. A transfervector is defined as a bacterial plasmid which contains a desired genedirectly 3′ to the polyhedrin promoter, as well as long viral sequencesflanking the promoter on the 5′ side. During cotransfection, homologousrecombination occurring between viral and transfer vector DNA willproduce a small percentage of viral genomes in which the polyhedrin genehas been replaced by the desired heterologous nucleic acid encoding thetarget polypeptide (0.1-5.0%). The wild-type progeny can bedifferentiated from the recombinant progeny by a conventional viralplaque assay. Recombinants in which the polyhedrin gene has beenreplaced, can be identified by their occlusion-negative plaque phenotypeobserved in a background of occlusion-positive wild-type plaques.

Because the polyhedrin gene is a non-essential gene for productive viralinfection, another advantage of baculovirus expression vectors is thatthe recombinants are viable, helper-independent viruses. Also,baculoviruses only infect Lepidopteran insects; thus, they arenoninfectious for vertebrates, and are, therefore, relatively safegenetic manipulation agents.

Notwithstanding the successes of BEVS and other systems for expressionof heterologous proteins in insect and mammalian cell culture,maintenance of the viability of transformed or transfected cell culturesremains a capricious undertaking. Many laboratories refer to tissueculture as a “black art,” due to the numerous variables that make itdifficult to determine solutions when problems arise. An intensive andtime-consuming systematic approach that examines the symptoms andmeticulously retraces each step in the culture process is usuallyrequired to identify the material or critical procedure that has createdthe viability issue. Problems such as poor cell growth and abnormalmorphology can result from materials that are poor quality,inappropriate, compromised, or contaminated and/or equipment that mustbe re-calibrated or re-setup to comply with manufacturer usage. Perhapsmost frustrating, cells of different lots may react differently tostandardized media and serum supplements resulting in unexpectedtoxicity or nutritional deficiency. Therefore, much of the time andexpense invested in preparation of protein expression vectors may belost when a protein production facility experiences difficulty inoptimizing cell culture protein production conditions. As such it wouldbe of great economic benefit to provide a generalized agent to a cellline to increase its viability, longevity and protein productioncapacity.

Insects, like other animals, have effective immune systems to combatboth biotic and abiotic foreign invasion. Interestingly, endoparasiticinsects spend a part of their life cycle inside the body of other insecthosts. Considerable effort has been expended investigating the mechanismby which these endoparasitic insects avoid the host immune system inthis parasitic relationship.

One well characterized parasitoid-host system in which there is immunesystem evasion is that of the endoparasitic wasp Campoletis sonorensisand its host, the tobacco budworm Heliothis virescens. In investigatinghow immunosuppression is regulated in this system, it became apparentthat a group of wasp viruses, known generically as polydnaviruses(PDVs), play a role in the suppression of the host immune system.Bracoviruses (BVs) and ichnoviruses (IVs) are the two main parasiticwasp associated PDVs. It is known that during oviposition, theendoparasitic insect, for example C. sonorensis, injects not only eggsbut also polydnavirus and oviduct proteins. Shortly thereafter, the hostinsect immune system begins to show evidence of altered activity and theendoparasitoid eggs remain free from encapsulation. The precisemechanism of this immune suppression is not presently understood.

The WHv1.0, WHv1.6 and VHv1.1 genes of C. sonorensis polydnavirus(CsPDV) have been cloned and sequenced. These genes are described asmembers of a polydnavirus “cysteine-rich” gene family. (Dib-Hajj et al.,Proc. Natl. Acad. Sci. (USA) 90: 3765 (1993)). It has been conjecturedthat these genes may play a role in preventing the recognition offoreign objects and/or the normal response of components of the immunesystem. (Summers et al., Proc. Natl. Acad. Sci. (USA) 92: 29 (1995)).Indeed, the VHv1.1 gene product of the C. sonorensis polydnavirus hasbeen implicated in the inhibition of the cellular immune response. This30 kDa protein is shown by indirect immunofluorescence to bind bothgranulocytes and plasmatocytes and is thought to inhibit encapsulation.(Li et al., J. Virol., 68: 7482 (1994)).

Recent PDV genome sequencing projects have revealed a novel family ofclosely related genes that exist in several genomes including, but notlimited to, the C. sonorensis IV (CsIV) Hyposoter fugitivus IV (HfIV),Glypta fumiferana IV (GfIV), Microplitis demolitor BV (MdBV), Cotesiacongregata BV (CcBV), Glyptapanteles indiensis BV (GiBV), and Toxoneuronnigriceps BV (TnBV) genomes. This family of genes has been namedvankyrins as their open reading frames (ORFs) encode proteins almostexclusively made up of ankyrin repeat domains. The PDV ankyrinrepeat-carrying proteins show significant identity to the ankyrinrepeats of the Iκβ family of transcription factor inhibitors suggestingthat they disrupt intracellular NF-κβ mediated signal transductioncascades known to play a role in both vertebrate and invertebrate immuneresponses. There are seven vankyrin ORFs encoded by the CsIV genome.

The inventors have discovered that the expression of two CsIV vankyrinsfrom a heterologous expression vector system increases the vitality,longevity, and therefore the protein productive capacity of cells inculture.

All references cited herein are hereby incorporated by reference intheir entirety for all purposes.

SUMMARY OF THE INVENTION

One aspect of the invention relates to a vankyrin expression vectorcomprising a nucleic acid encoding the polypeptide of SEQ ID NO: 2.Another aspect of the invention relates to a vankyrin expression vectorcomprising a nucleic acid encoding the polypeptide of SEQ ID NO: 4. Yetanother aspect of the invention relates to a vankyrin expression vectorcomprising a nucleic acid that hybridizes to the nucleic acid of SEQ IDNO: 1 under stringent conditions wherein the nucleic acid encodes apolypeptide capable of enhancing longevity and/or protein production ofa cell line in which it is expressed. A further aspect of the inventionrelates to a vankyrin expression vector comprising a nucleic acid thathybridizes to the nucleic acid of SEQ ID NO: 3 under stringentconditions wherein the nucleic acid encodes a polypeptide capable ofenhancing longevity and/or protein production of a cell line in which itis expressed. Another aspect of the invention relates to a vankyrinexpression vector comprising a nucleic acid of SEQ ID NO: 1. Yet anotheraspect relates to a vankyrin expression vector comprising a nucleic acidof SEQ ID NO: 3.

Another aspect of the invention relates to a recombinant cell comprisinga first nucleic acid selected from the group consisting of a nucleicacid encoding the polypeptide of SEQ ID NO: 2 a nucleic acid thathybridizes to the nucleic acid of SEQ ID NO: 1 under stringentconditions wherein the nucleic acid encodes a polypeptide capable ofenhancing longevity and/or protein production of a cell line in which itis expressed; and a nucleic acid of SEQ ID NO: 1; and/or a secondnucleic acid selected from the group consisting of: a nucleic acidencoding the polypeptide of SEQ ID NO: 4; a nucleic acid that hybridizesto the nucleic acid of SEQ ID NO: 3 under stringent conditions whereinthe nucleic acid encodes a polypeptide capable of enhancing longevityand/or protein production of a cell line in which it is expressed; and anucleic acid of SEQ ID NO: 3.

Another aspect of the invention relates to a method of enhancing targetprotein production of a cell line producing a target protein comprisingtransforming cells of the cell line with a vankyrin expression vector,growing the cell line; and isolating the target protein from the cellline.

Another aspect of the invention relates to a method of generating arecombinant cell line capable of enhanced target protein productioncomprising transforming a cell line with a heterologous nucleic acidencoding and driving the expression of a target protein; andtransforming cells of the cell line with a vankyrin expression.

Yet another aspect of the invention relates to a method of generating arecombinant target protein-producing cell line capable of enhancedtarget protein production comprising constructing a vankyrin expressionvector and transforming cells of the cell line with the vankyrinexpression vector.

Another aspect of the invention relates to a method of enhancinglongevity of a cell line producing a target protein comprisingtransforming cells of the cell line with a vankyrin expression vector,to obtain a transformed cell line producing a target protein; growingthe transformed cell line producing a target protein longer than a thecell line not transformed with the vector.

Additional advantages of the present invention will become readilyapparent to those skilled in this art from the following detaileddescription, wherein only the preferred embodiment of the invention isshown and described, simply by way of illustration of the best modecontemplated of carrying out the invention. As will be realized, theinvention is capable of other and different embodiments, and its severaldetails are capable of modifications in various obvious respects, allwithout departing from the invention. The present invention may bepracticed without some or all of these specific details. In otherinstances, well known process operations have not been described indetail, in order not to unnecessarily obscure the present invention.Accordingly, the drawings and description are to be regarded asillustrative in nature, and not as restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Morphology of 4-day post infected (4 d p.i.) Sf9 (panel A) andS1-2 (panel B) cells exposed to recombinant AcMNPV's expressing CsIVvankyrin proteins. Cells infected with recombinant viruses expressingfat body specific P-ank-1 and I²-ank-3 proteins (asterisks) are morestable and resemble non-infected cells at 4 d p.i. Cells exposed torecombinant viruses expressing the remaining CsIV genes undergoapoptosis and lysis by 4 d p.i. and resemble cells infected with wildtype AcMNPV. 40× magnification.

FIG. 2. Protein expression is extended in Sf9 cells infected withrecombinant AcMNPV expressing Fat-Body specific CsIV ankyrin genesP-ank-1 and I²-ank-3. Western blots represent detection of proteins infreshly overlayed culture media presented to cells at each subsequentday following infection. The CsIV vankyrin genes are intracellularproteins and lack secretory signals, thus protein detected in the mediaoverlay is the result of that released by cell lysis or rupturefollowing infection. Delayed detection of proteins from P-ank-1 andI²-ank-3 viruses until day 3 p.i. is resultant of the enhanced longevityof Sf9 cells occurring early during infection by these viruses (asevidenced in FIG. 1).

FIG. 3. The cDNA and amino acid sequences of P-ank-1 (SEQ ID NO: 1 and3, respectively) and I²-ank-3 (SEQ ID NO: 2 and 4, respectively).

FIG. 4. Morphology of Sf9 cells exposed to recombinant AcMNPV'sexpressing CsIV vankyrin proteins over time. Cells infected withrecombinant viruses expressing fat body specific P-ank-1 and I²-ank-3proteins (asterisks) are more stable and increase the longevity of thecells through 6 days (6D) post infection such that they resemblenon-transfected control cells. Cells exposed to recombinant virusesexpressing the remaining CsIV genes undergo apoptosis and lysis by 4 dp.i. and resemble cells infected with wild type AcMNPV. 40×magnification.

FIG. 5. Morphology of S1-2 cells exposed to recombinant AcMNPV'sexpressing CsIV vankyrin proteins over time. Cells infected withrecombinant viruses expressing fat body specific P-ank-1 and I²-ank-3proteins (asterisks) are more stable and maintain the vitality of thecells through 6 days (6D) post infection such that they resemblenon-transfected control cells. Cells exposed to recombinant virusesexpressing the remaining CsIV genes undergo apoptosis and lysis by 4 dp.i. and resemble cells infected with wild type AcMNPV. 40×magnification.

FIG. 6. Shows the yield of recombinant vankyrin protein produced incells infected by different recombinant AcMNPVs. A cell line wasinfected with different recombinant AcMNPVs encoding different vankyrinproteins. Next, the vankyrin protein encoded by the each differingrecombinant AcMNPV was isolated and quantified. The inventors note thatcells infected by recombinant AcMNPVs encoding P-ank-1 and I²-ank-3produced significantly larger quantities of their encoded CsIV vankyrinproteins, i.e., P-ank-1 and I²-ank-3 protein, respectively, than cellstransgenically expressing the other CsIV vankyrin proteins.

DETAILED DESCRIPTION OF THE INVENTION

It is an object of the current invention to provide methods andcompositions relating to the expression of vankyrin proteins in celllines to increase their viability, longevity and capacity for proteinproduction. The vankyrin gene family comprises 7 genes on CsIV genomesegments P and I². Each vankyrin gene encodes an open reading frame ofabout 500-bp possessing 4 ankyrin repeat protein motifs. The vankyrinprotein motifs show significant identities to ankyrin motifs of Cactus,the Drosophila IκB protein. MdBV and CsIV vankyrin genes align with the4 C-terminal ankyrin repeat domains of IκBs but lack N-terminal repeatsthat function to mask nuclear localization signals (NLS) and sequesteruninduced NF-κB dimers in the cytoplasm.

The seven vankyrin genes are I²-ank-1, I²-ank-2, I²-ank-3, P-ank-1,P-ank-2, P-ank-3 and P-ank-4. The inventors have discovered that theexpression of P-ank-1 and I²-ank-3 proteins in cell culture hasincreased the cells' viability, longevity and, therefore, capacity forendogenous and/or heterologous target protein production. Specifically,the present invention relates to the enhanced expression of endogenousand/or heterologous target proteins/polypeptides in recombinant cellsthat are also expressing P-ank-1 and/or I²-ank-3 protein compared toexpression host cells that are not expressing P-ank-1 and/or I²-ank-3protein.

Before describing the invention in greater detail the followingdefinitions are set forth to illustrate and define the meaning and scopeof the terms used to describe the invention herein:

The term “nucleic acid molecule” is meant to include DNA, RNA and mixedDNA-RNA sequences. In addition to the typically found A, T, U, G and Cresidues, a nucleic acid molecule may also include related residues suchas, for example, inosine (I).

The term “polynucleotide” or “oligonucleotide” as used herein refers toa polymeric form of nucleotides of any length, either ribonucleotides ordeoxyribonucleotides. This term refers only to the primary structure ofthe molecule. Thus, this term includes double and single stranded DNA,triplex DNA, as well as double and single stranded RNA. It also includesmodified, for example, by methylation and/or by capping, and unmodifiedforms of the polynucleotide.

The term “promoter region” refers to a DNA sequence that functions tocontrol the transcription of one or more nucleic acid sequences, locatedupstream with respect to the direction of transcription of thetranscription initiation site of the gene, and is structurallyidentified by the presence of a binding site for DNA-dependent RNApolymerase, transcription initiation sites and any other DNA sequences,including, but not limited to transcription factor binding sites,repressor and activator protein binding sites, calcium or cAMPresponsive sites, and any other nucleotide sequences known to actdirectly or indirectly to regulate transcription from the promoter.

The term “heterologous DNA” or “heterologous RNA” refers to DNA or RNAthat does not occur naturally as part of the genome or DNA or RNAsequence in which it is present, or in which it is found, a cell orlocation or locations in the genome or DNA or RNA sequence that differsfrom that which it is in found in nature. Heterologous DNA or RNA is notendogenous to the cell into which it is introduced, but has beenobtained from another cell or synthetically or recombinantly produced.Generally, though not necessarily, such DNA encodes RNA and protein notnormally produced by the cell in which the DNA is transcribed orexpressed. Similarly exogenous RNA encodes protein not normallyexpressed in the cell in which the exogenous RNA is present.Heterologous DNA or RNA may also be referred to as foreign DNA or RNA.Any DNA or RNA that one of skill in the art would recognize asheterologous or foreign to the cell in which it is expressed is hereinencompassed by the term heterologous DNA or heterologous RNA. Examplesof heterologous DNA include, but are not limited to, DNA that encodes aprotein, polypeptide, reporter nucleic acid sequence, transcriptional ortranslational regulatory sequences, selectable or traceable markerprotein, such as a protein that confers drug resistance, RNA includingmRNA and antisense RNA, and ribozymes.

The term “recombinant polynucleotide” as used herein refers to apolynucleotide of genomic, cDNA, semisynthetic or synthetic originwhich, by virtue of its origin or manipulation: (1) is not associatedwith all or a portion of the polynucleotide with which it is associatedin nature and/or (2) is linked to a polynucleotide other than that towhich it is linked in nature. The term “cDNA” or “complementary DNA”refers to single stranded or double stranded DNA sequences obtained byreverse transcription of messenger RNA isolated from a donor cell. Forexample, treatment of messenger RNA with a reverse transcriptase such asAMV reverse transcriptase or M-MuLV reverse transcriptase in thepresence of an oligonucleotide primer will furnish an RNA-DNA duplexwhich can be treated with RNase H, DNA polymerase and DNA ligase togenerate double stranded cDNA. If desired, the double stranded cDNA canbe denatured by conventional techniques such as shearing to generatesingle stranded cDNA.

The term “operably linked” refers to the linkage of a DNA segment toanother DNA segment in such a way as to allow the segments to functionin their intended manners. A DNA sequence encoding a gene product isoperably linked to a regulatory sequence when it is ligated to theregulatory sequence, such as, for example, promoters, enhancers and/orsilencers, in a manner which allows modulation of transcription of theDNA sequence, directly or indirectly. For example, a DNA sequence isoperably linked to a promoter when it is ligated to the promoterdownstream with respect to the transcription initiation site of thepromoter, in the correct reading frame with respect to the transcriptioninitiation site, and allows transcription elongation to proceed throughthe DNA sequence. An enhancer or silencer is operably linked to a DNAsequence coding for a gene product when it is ligated to the DNAsequence in such a manner as to increase or decrease, respectively, thetranscription of the DNA sequence. Enhancers and silencers may belocated upstream, downstream or embedded within the coding regions ofthe DNA sequence. A DNA for a signal sequence is operably linked to DNAcoding for a polypeptide if the signal sequence is expressed as apreprotein that participates in the secretion of the polypeptide.Linkage of DNA sequences to regulatory sequences is typicallyaccomplished by ligation at suitable restriction sites or via adaptersor linkers inserted in the sequence using restriction endonucleasesknown to one of skill in the art.

The term “target” protein or polypeptide, refers to a protein ofinterest that is expressed in the recombinant cells also expressingP-ank-1 and/or I²-ank-3 protein. Preferably, the recombinant cell isused as bioreactor for the production of the target protein. The targetprotein may be an endogenous protein naturally produced by the host celltype. For example, if the host cell type is a hybridoma, the targetprotein may be a monoclonal antibody. Alternatively, the target proteincan be encoded by a heterologous recombinant nucleic acid, e.g. a cDNA.In this case, the target protein will be a heterologous protein, i.e.,one that is not naturally expressed by the host cell line.

Central to the invention is the “vankyrin expression vector.” A vankyrinexpression vector is any genetic element, e.g., a plasmid, chromosome,virus, capable of bringing about the expression of a P-ank-1 (SEQ ID NO:2) and/or I²-ank-3 (SEQ NO: 4) proteins or proteins substantiallysimilar thereto, i.e., those having similar amino acid sequences and thesame functionalities with regard to the ability to provide enhanced celllongevity and/or protein productive capacity. Preferably, proteinsP-ank-1 (SEQ ID NO: 2) and I²-ank-3 (SEQ NO: 4) are encoded by SEQ IDNO: 1 and SEQ NO: 3, respectively. The skilled artisan will alsoappreciate that invention also encompasses vankyrin expression vectorsequences comprising sequences substantially identical to SEQ ID NOs: 1and 3. Such sequences may differ from SEQ ID NOs: 1 and 3, respectively,with regard to the identity of at least one nucleotide base.

However, all polynucleotides sequences “substantially identical” to SEQID NOs: 1 and 3 hybridize under stringent conditions (as defined herein)to all or a portion of the complements of SEQ ID NOs: 1 and 3 (i.e.,target sequences), respectively. The terms “hybridize(s) specifically”or “specifically hybridize(s)” refer to complementary hybridizationbetween an oligonucleotide (e.g., a primer or labeled probe) and atarget sequence. The term specifically embraces minor mismatches thatcan be accommodated by reducing the stringency of the hybridizationmedia to achieve the desired priming for the PCR polymerases ordetection of hybridization signal.

Under stringent hybridization conditions, only highly complementary,i.e., substantially identical nucleic acid sequences, hybridize.Preferably, such conditions prevent hybridization of nucleic acidshaving 3 or more mismatches out of 20 contiguous nucleotides, morepreferably 2 or more mismatches out of 20 contiguous nucleotides, mostpreferably one or more mismatch out of 20 contiguous nucleotides. Thehybridizing portion of the hybridizing nucleic acid is at least about90%, preferably at least about 95%, or most preferably about at leastabout 98%, identical to the sequence of a target sequence, or itscomplement.

Hybridization of a nucleic acid to a nucleic acid sample under stringentconditions is defined below. Nucleic acid duplex or hybrid stability isexpressed as a melting temperature (T_(m)), which is the temperature atwhich the probe dissociates from the target DNA. This meltingtemperature is used to define the required stringency conditions. Ifsequences are to be identified that are substantially identical to theprobe, rather than identical, then it is useful to first establish thelowest temperature at which only homologous hybridization occurs with aparticular concentration of salt (e.g. SSC or SSPE). Then assuming that1% mismatching results in a 1° C. decrease in T_(m), the temperature ofthe final wash in the hybridization reaction is reduced accordingly (forexample, if sequences having >95% identity with the probe are sought,the final wash temperature is decrease by 5° C.). In practice, thechange in T_(m) can be between 0.5° C. and 1.5° C. per 1% mismatch.

Stringent conditions involve hybridizing at 68° C. in 5×SSC/5×Denhart'ssolution/1.0% SDS, and washing in 0.2×SSC/0.1% SDS at room temperature.Moderately stringent conditions include washing in 3×SSC at 42° C.Additional guidance regarding such conditions is readily available inthe art, for example, Sambrook, Fischer and Maniatis, Molecular Cloning,a laboratory manual, (2nd ed.), Cold Spring Harbor Laboratory Press, NewYork, (1989) and F. M. Ausubel et al eds., Current Protocols inMolecular Biology, John Wiley and Sons (1994).

Moreover, vankyrin expression vector containing polynucleotides“substantially similar or identical” to SEQ ID NO: 1 and 3,respectively, are capable of providing a cell line with enhancedlongevity and/or protein production capability. In one embodiment, whenexpressed in Sf9 or S1-2 cells, polynucleotides “substantially similaror identical” to SEQ ID NO: 1 and 3, respectively, are capable ofimproving the longevity of those cells to such an extent that theyresemble non-transfected Sf9 or S1-2 cells, whereas Sf9 or S1-2 cellstransfected with a vector not containing polynucleotides “substantiallysimilar or identical” to SEQ ID NO: 1 and 3, respectively. Preferably,the vector is a baculovirus vector. In another embodiment,polynucleotides “substantially similar or identical” to SEQ ID NO: 1 and3, respectively, are capable of providing enhanced protein production ina cell line in which they are expressed, relative to a host cell linetransfected in a similar manner by a vector lacking suchpolynucleotides. For example, polynucleotides “substantially similar oridentical” to SEQ ID NO: 1 and 3, respectively, when expressed in Sf9 orS1-2 cells, are capable providing enhanced protein production in thosecells, relative to Sf9 or S1-2 cells transfected with a vector notcontaining polynucleotides “substantially similar or identical” to SEQID NO: 1 and 3, respectively.

The vankyrin expression vector contains sequences to facilitateexpression of P-ank-1 and/or I²-ank-3 proteins in the host cell. Suchsequences differ depending on the host organism; they include promotersequences, for example but not limited to a polyhedrin promoter, SV40promoter, or a conditionally activated promoter such as ametallothionein promoter to effect transcription; enhancer sequences toincrease transcription; ribosomal binding site sequences; andtranscription and translation termination sequences. The vector may alsooptionally behave either as an autonomous unit of polynucleotidereplication within a cell (i.e., capable of replication under its owncontrol) or being rendered capable of replication by insertion into ahost cell chromosome, having attached to it another polynucleotidesegment, so as to bring about the replication. Suitable vectors include,but are not limited to, viruses, plasmids, bacteriophages, yeastartificial chromosomes (YACs), cosmids, and the like. Vectors maycontain polynucleotide sequences which are necessary to effect ligationor insertion of the vector into a desired host cell and the expressionof its coding region(s). Additionally, the vankyrin expression vectoritself may also contain heterologous nucleic acids encoding and drivingthe expression of target heterologous proteins and/or reporter proteins.

The skilled artisan will recognize that a wide range of vectors may beconstructed to permanently, constitutively, conditionally or transientlydrive P-ank-1 and/or I²-ank-3 expression in a wide range of insect andmammalian cell lines. The skilled artisan would know how to operablylink the aforementioned sequences. It is to be understood that thisinvention is intended to include other forms of expression vectors, hostcells and transformation techniques which serve equivalent functions andwhich become known to the art hereto.

The preferred “vankyrin expression vector” is part of a baculovirusexpression system engineered to express P-ank-1 and/or I²-ank-3 proteinsand the control of a polyhedrin promoter in the cells it infects. Most,preferably, the baculovirus is an Autographa californica baculovirus(AcNPV), and expression of P-ank-1 and/or I²-ank-3 protein is driven bya polyhedrin promoter. The baculovirus Autographa californica mononuclear polyhedrosis virus (AcMNPV), used in the examples as theoriginal source of viral DNA was isolated according to proceduresdescribed in G. E. Smith and M. D. Summers, Virology, 89:517-527 (1978)and G. E. Smith and M. D. Summers, J. Virol., 39:125-137 (1981).According to the preferred embodiment of this invention, a particularstrain of AcMNPV, E2, is utilized. However, those skilled in the art whohave the benefit of this disclosure will recognize that otherbaculoviruses and other baculovirus strains may also be suitablyutilized to obtain viral DNA. In particular, it is expected that atleast the closely related and naturally occurring strains, Trichoplusiani MNPV, Rachiplusia ou MNPV, Galleria mellonella MNPV and anyplaque-purified strains such as the M3′ R9, S1 and S3 strains of AcMNPVisolated and characterized in G. E. Smith and M. D. Summers, J. Virol.,33:311-319 (1980), as well as Bombyx mori NPV (BmNPV) may be utilized toadvantage. Further description of those and other strains are found inG. E. Smith and M. D. Summers, Virol., 89:517-527 (1978).

Plasmids for the aforementioned BEVS carrying SEQ ID NO: 1 and/or 3, orsequences substantially similar thereto, may be designed according toconventional techniques known in the art and as described in M. D.Summers and G. E. Smith, A Manual of Methods for Baculovirus Vectors andInsect Cell Culture Procedures, Texas Agricultural Experiment StationBulletin No. 1555, Texas A&M University (1987) (“Bulletin No. 1555”).(See also V. A. Luckow and M. D. Summers, Virol., 170:31-39 (1989)). Inthe preferred embodiment, the Baculovirus Expression Vector System fromBD Biosciences Pharmingen is used which employs a modified Autographacalifornica nuclear polyhedrosis virus (AcNPV) genome—BD BaculoGold™DNA, and an appropriate transfer vector. The diversity of AcNPV-basedtransfer vectors, combined with available S. frugiperda Sf9 and Sf21cell lines, establish baculovirus expression as a preferred system forfunctional eukaryotic gene expression and the large-scale production ofrecombinant proteins.

Although the methodology described herein is believed to containsufficient detail to enable one skilled in the art to practice thepresent invention, the plasmids can be constructed and purified usingstandard recombinant DNA techniques described in T. Maniatis, E. F.Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory (1982) under the current regulations describedin United States Dept. of HEW, National Institute of Health (NIH)Guidelines for Recombinant DNA Research. These references includeprocedures for the following standard methods: cloning procedures withE. coli plasmids, transformation of E. coli cells, plasmid DNApurification, phenol extraction of DNA, ethanol precipitation of DNA,agarose gel electrophoresis, purification of DNA fragments from agarosegels, and restriction endonuclease and other DNA-modifying enzymereactions. Accordingly, these available references are incorporatedherein by reference.

A “reporter nucleic acid sequence” is a DNA molecule that expresses adetectable gene product, which may be reporter RNA or reporter protein.The detection may be accomplished by any method known to one of skill inthe art. For example, detection of mRNA expression may be accomplishedby using Northern blot analysis and detection of protein may beaccomplished by staining with antibodies specific to the protein, e.g.Western blot analysis. Preferred reporter nucleic acid sequences arethose that are readily detectable. A reporter nucleic acid sequence maybe operably linked in a DNA construct with a regulatory DNA sequencesuch that detection of the reporter nucleic acid sequence productprovides a measure of the transcriptional activity of the regulatorysequence. Examples of reporter nucleic acid sequences include, but arenot limited to, those coding for alkaline phosphatase, chloramphenicolacetyl transferase (CAT), luciferase, beta-galactosidase and alkalinephosphatase.

The terms “transformed” or “transfected” are used interchangeably andrefer to the process by which exogenous DNA or RNA is transferred orintroduced into an appropriate host cell. Additionally, nucleic acidsencoding other heterologous proteins may be introduced into the hostcell. Such transfected cells include stably transfected cells whereinthe inserted DNA is rendered capable of replication in the host cell.Typically, stable transfection requires that the exogenous DNA betransferred along with a selectable marker nucleic acid sequence, suchas for example, a nucleic acid sequence that confers antibioticresistance, which enables the selection of the stable transfectants.This marker nucleic acid sequence may be ligated to the exogenous DNA orbe provided independently by simultaneous cotransfection along with theexogenous DNA. Transfected cells also include transiently expressingcells that are capable of expressing the RNA or DNA for limited periodsof time. The transfection procedure depends on the host cell beingtransfected. It can include packaging the polynucleotide in a virus aswell as direct uptake of the polynucleotide. Transformation can resultin incorporation of the inserted DNA into the genome of the host cell orthe maintenance of the inserted DNA within the host cell in plasmidform. Methods of transformation/transfection are well known in the artand include, but are not limited to, direct injection, such asmicroinjection, viral infection, particularly replication-deficientadenovirus infection, electroporation, lipofection, calciumphosphate-mediated direct uptake and the like.

The term “host cell” generally refers to eukaryotic cells and includesany transformable cell which is capable of expressing a P-ank-1 and/orI²-ank-3 proteins and can be, or has been, used as a recipient for avankyrin expression vector. Once cells have transiently or stably takenup the vankyrin expression vector they are “recombinant” cells. DNA iscommonly transferred or introduced into recipient mammal cells bycalcium phosphate-mediated gene transfer, electroporation, lipofection,viral infection and the like. General methods, vectors and generalconsiderations for gene transfer and expression may be found in M.Kriegler, Gene Transfer and Expression: A Laboratory Manual, StocktonPress (1990). Direct gene transfer to cells in vivo is achieved by theuse of modified viral vectors, including retroviruses, adenoviruses,adeno-associated viruses and herpes viruses, liposomes, and directinjection of DNA into certain cell types. See, e.g., Wilson, Nature,365: 691-692 (1993); Plautz et al, Annals. NY Acad. Sci., 716: 144-153(1994); Farhood et al, Annals NY Acad. Sci., 716: 23-34 (1994) and Hydeet al Nature, 362: 250-255 (1993).

Recombinant cells provided by this invention expressing P-ank-1 and/orI²-ank-3 proteins are intended to produce target polypeptides,preferably human proteins and fragments thereof. The process involvesculturing the recombinant cells under conditions wherein the endogenousor heterologous target proteins are expressed, e.g., by inducing theactivity of a conditional promoter, and purifying the target proteinfrom the cell culture. Purification of target proteins is within theskill set or the skilled artisan and generally involves the steps ofcell lysis, homogenization, centrifugation and separation of the desiredprotein by processes such as salt fractionation, precipitation, and avariety of chromatographic methods such as anion exchangechromatography, hydrophobic interaction chromatography, high resolutionchromatography, gel filtration chromatography and the like.

One aspect of this invention, relates to cells transiently expressing avankyrin expression vector. In one embodiment of this aspect of theinvention, the transient expression of the P-ank-1 and/or I²-ank-3proteins serves to temporarily strengthen the vitality of the cultureexpressing them. It is envisaged that this temporary increase invitality will allow for the increased production of target proteinsproduced by and harvested from the host cell line. For example, anestablished monoclonal antibody (Mab)-producing hybridoma cell line maybe transiently transfected with the vankyrin expression element toobtain an increase in antibody production. The most simple method for invitro production of Mabs is standard tissue culture in either largeflasks or roller bottles. The production of Mab by hybridomas in tissueculture is hybridoma-dependent and can vary between 1-100 μg/ml.Therefore, it is often necessity to concentrate Mab from supernatant.Transfecting a hybridoma with the vankyrin expression element will allowfor increased Mab production and lessen the need for a technician toconcentrate antibody in the supernatant.

In another embodiment of this aspect of the invention, the cellstransiently transfected with a vankyrin expression vector are alsotransiently or permanently co-transfected with an additional expressionelement having a heterologous nucleic acid sequence encoding and drivingthe expression of a heterologous target protein.

Another aspect of the invention relates to cells in which a vankyrinexpression element is stably integrated into the cells' genome, thusrendering a recombinant cell line that provides superior proteinproductive capacity when compared to its wild type cell counterpart. Inone embodiment of this aspect of the invention, such a cell line isamenable to further permanent transfection with an additional expressionvector carrying a nucleic acid sequence encoding a target protein ofinterest. In another embodiment, such a cell line is amenable totransient transfection with an additional expression vector carrying anucleic acid sequence encoding a target protein of interest. In anotherembodiment, the target proteins produced by and harvested from the cellshaving permanently integrated vankyrin expression vectors may beproteins endogenously produced by the host cells themselves.

Yet a further aspect of the invention relates to a vankyrin expressionvector that contains additional nucleic acid sequences encoding one ormore heterologous target proteins of interest. Such a vector could bepermanently or transiently introduced into a host cell line.

The recombinant cells having the “vankyrin expression vector” expressingP-ank-1 and/or I²-ank-3 proteins are mammalian, such as, but not limitedto Chinese hamster ovary (CHO) cells, COS-7 cells, fibroblasts as wellas C127, 3T3, CHO, HeLa and BHK cell lines. Most preferably, the cellsare insect cells such as, but not limited to S2 cells, Schneider cells,S12 cells, 5B1-4, Tn5, and Sf9 cells. The Spodoptera frugiperda Sf9 cellline may be obtained from American Type Culture Collection (Rockville,Md.) and is assigned accession number ATCC CRL 1711. See M. D. Summersand G. E. Smith, Bulletin No. 1555, suora. Those skilled in the art whohave the benefit of this disclosure will recognize that other clonalderivatives of the Sf9 cell line as well as Trichoplusia ni and otherinsects such as the silkworm, Bombyx mori, or insect cell culturesderived there from can be used to advantage.

The standard methods of insect cell culture, cotransfection andpreparation of plasmids in accordance with the examples, are set forthin M. D. Summers and G. E. Smith, A Manual of Methods for BaculovirusVectors and Insect Cell Culture Procedures, Texas AgriculturalExperiment Station Bulletin No. 1555, Texas A&M University (1987). Thisreference also pertains to the standard methods of cloning genes intoAcMNPV transfer vectors, plasmid DNA isolation, transferring genes intothe AcMNPV genome, viral DNA purification, radiolabelling recombinantproteins and preparation of insect cell culture media. Accordingly, thisavailable reference is incorporated herein by reference.

The procedures for the cultivation of viruses and cells are described inL. E. Volkman and M. D. Summers, J. Virol, 19:820-832 (1975) and L. E.Volkman, M. D. Summers and C. H. Hsieh, J. Virol, 19:820-832 (1976).Viral growth kinetics were determined as described by L. E. Volkman, etal., suora, using S. frugiperda and a 1.5% agarose overlay.

Example 1

For example, when expressed in Sf9 or S1-2 cells, polynucleotides“substantially similar or identical” to SEQ ID NO: 1 and 3,respectively, are capable improving the longevity of those cells to suchan extent that they resemble non-transfected Sf9 or S1-2 cells, whereasSf9 or S1-2 cells transfected with a vector not containingpolynucleotides “substantially similar or identical” to SEQ ID NO: 1 and3, respectively, rapidly lyse about 4 days post infection. FIG. 1.Moreover, the longevity of a cell line expressing polynucleotides“substantially similar or identical” to SEQ ID NO: 1 and 3,respectively, continues to be maintained such that it resemblesnon-infected cells at about 6 to about 7 days post infection. FIGS. 2, 4and 5. Therefore, when an Sf9 or S1-2 cell line is infected with aAcMNPV comprising polynucleotides “substantially similar or identical”to SEQ ID NO: 1 or 3, respectively, the infected Sf9 or S1-2 cell linehas enhanced longevity relative to an Sf9 or S1-2 cell line infectedwith a wild type AcMNPV.

Example 2

Polynucleotides “substantially similar or identical” to SEQ ID NO: 1 and3, respectively, are capable of providing enhanced protein production ina cell line in which they are expressed, relative to a host cell linetransfected in a similar manner by a vector lacking suchpolynucleotides. Specifically, polynucleotides “substantially similar oridentical” to SEQ ID NO: 1 and 3, respectively, when expressed in Sf9 orS1-2 cells, are capable providing enhanced protein production in thosecells, relative to Sf9 or S1-2 cells transfected with a vector notcontaining polynucleotides “substantially similar or identical” to SEQID NO: 1 and 3, respectively. As can be seen in FIG. 2, proteinexpression is extended in Sf9 cells infected with recombinant AcMNPVexpressing CsIV ankyrin genes P-ank-1 and I²-ank-3. Western blots inFIG. 2, represent detection of proteins in freshly overlayed culturemedia presented to cells at each subsequent day following infection. TheCsIV vankyrin genes are intracellular proteins and lack secretorysignals, thus protein detected in the media overlay is the result ofthat released by cell lysis or rupture following infection. Delayeddetection of proteins from P-ank-1 and I²-ank-3 viruses until day 3 postinfection is resultant of the enhanced longevity of Sf9 cells occurringearly during infection by these viruses (as evidenced in FIG. 1).Additionally, because cells expressing polynucleotides “substantiallysimilar or identical” to SEQ ID NO: 1 and 3, respectively, are able toproduce proteins for a longer period of time, they are able to producemore protein in total, thus providing an enhanced protein productioncapability. Therefore, when an Sf9 or S1-2 cell line is infected with aAcMNPV comprising polynucleotides “substantially similar or identical”to SEQ ID NO: 1 or 3, respectively, the infected Sf9 or S1-2 cell linehas enhanced protein production relative to an Sf9 or S1-2 cell lineinfected with a wild type AcMNPV.

1-10. (canceled)
 11. A recombinant cell comprising a first nucleic acidselected from the group consisting of: a. a nucleic acid encoding thepolypeptide of SEQ ID NO: 2; b. a nucleic acid that hybridizes to thenucleic acid of SEQ ID NO: 1 under stringent conditions wherein thenucleic acid encodes a polypeptide capable of enhancing longevity and/orprotein production of a cell line in which it is expressed; and c. anucleic acid of SEQ ID NO: 1; and/or a second nucleic acid selected fromthe group consisting of: a. a nucleic acid encoding the polypeptide ofSEQ ID NO: 4; b. a nucleic acid that hybridizes to the nucleic acid ofSEQ ID NO: 3 under stringent conditions wherein the nucleic acid encodesa polypeptide capable of enhancing longevity and/or protein productionof a cell line in which it is expressed; and c. a nucleic acid of SEQ IDNO:
 3. 12. The cell of claim 11 wherein said cell is an Sf9 or an S12insect cell.
 13. The cell of claim 11 wherein said vector is abaculovirus expression vector.
 14. The cell of claim 11 wherein thefirst and/or second nucleic acid are stably transfected.
 15. The cell ofclaim 11 wherein said cell expresses a target protein.
 16. The cell ofclaim 15, further comprising a heterologous nucleotide encoding saidtarget protein.
 17. The cell of claim 15 wherein said target protein isan endogenous protein.
 18. The cell of claim 17, wherein said cell is ahybridoma and said target protein is a monoclonal antibody. 19-22.(canceled)